Such methods for the optical, especially photometric, determining of a pH value or a concentration of an analyte in the measured medium are known. Frequently, in such case, an indicator is used, whose absorption spectrum, or emission spectrum, as the case may be, in the case of excitation with a certain wavelength, changes as a function of the measured variable to be determined.
In the case of photometric determination of such measured variables, a number of disturbing factors can degrade the accuracy of measurement. For assuring a high accuracy of measurement, consequently, referencing the measurement signals is of great importance. From DE 103 16 685 A1, for example, a measuring apparatus for the photometric measuring of concentration of a chemical substance in a measured solution is known, in the case of which a transmitting unit produces electromagnetic radiation in at least two wavelength ranges and radiates into a cuvette containing the measured solution, wherein the electromagnetic radiation in a first wavelength range serves for measurement purposes and the electromagnetic radiation in a second wavelength range is taken into consideration for reference purposes. In such case, the electromagnetic radiation in both wavelength ranges takes the same path through the cuvette and the measured solution. This is achieved with a dual light emitting diode as light source, which is operated in such a manner, that it alternately transmits electromagnetic radiation in the two wavelength ranges. Disturbing influences, such as clouding of the measured solution or impurities dissolved in the measured solution, or refraction and reflection at interfaces, e.g. on windows, within the beam paths of both beams should be eliminated in this way.
From EP 850 409 B1, a method is known, in the case of which an analyte sensitive indicator brought in contact with the measured medium is excited simultaneously with activation of two modulated light signals of different wavelength. The wavelengths of the light signals are selected from two different regions of the excitation spectrum of the indicator, which are influenced by the concentration of the analyte to be detected in different manners. The corresponding signals emitted by the indicator are detected, demodulated and, from the intensity ratio of the demodulated emission signals, the concentration of the analyte is determined.
Such methods, in the case of which the ratio of the intensity of a signal influenced by the measured variable and the intensity of a signal not influenced, or influenced in different measure, by the measured variable is formed, are referred to as ratiometric methods. In the case of these methods, a lessening of the indicator concentration in the measuring path (a so-called washing out or “leaching”) and a bleaching by photochemical reactions (so-called photobleaching or “bleaching”) do not, due to the ratio formation between the intensities of the two signals, affect the measurement result.
Since, in the case of conventional ratiometry, the two signals are always separately registered, the known methods require two separated signal paths or else means, which permit a sequential registering of the signals. Alternatively, as described in EP 850 409 B1, the separation of the two signals can also occur by modulating the two radiated signals and then demodulating for the evaluation. This is associated with corresponding apparatus complexity and/or space requirements.
In the article “Dual wavelength referencing of optical fiber sensors” by G. Murtaza, J. M. Senior, Optics Communications, 120 (1995), Pgs. 348-357, the application of ratiometric methods in sensors with optical fibers is considered. In the article, it is noted that the measurement accuracy of the method is limited by the fact that two light signals of different wavelengths react in different manners to environmental influences, wherein especially two light signals of different wavelengths are transmitted in different measure by optical components. A quantifying of this effect or proposals for the solution of this problem are not given.
In EP 1000345 B1 and in the article “Dual Lifetime Referencing (DLR)—a New Scheme for Domain Information” in “New Trends in fluorescence spectroscopy: application to chemical and life science”, B. Valeur, J.-C. Brochon (editors), Springer Verlag (publisher), Berlin Heidelberg 2001, Pgs. 257-274, a photometric method for determining a measured variable in a sample, especially the pH value or a concentration of an analyte, is given. In such case, two indicators are excited with a signal of a single wavelength to luminesce, wherein the luminescence intensity of the one indicator changes as a function of the measured variable, while the luminescence intensity of the other indicator, which serves as reference indicator, is not influenced by the measured variable. Moreover, the indicators are so selected, that the decay time of the luminescence of the reference indicator is significantly longer than that of the indicator influenced by the measured variable. If the intensity of the exciter signal is periodically modulated, there results, due to the different decay times between the two luminescence signal intensities of the measured value sensitive indicator and the reference indicator, a phase difference. The total intensity, i.e. the sum of the luminescence signals, has, relative to a reference (for example, the periodic exciter signal), a phase difference, which is dependent on the intensity ratio of the luminescence signals of the two indicators, and, thus, serves as a measure for the measured variable to be determined.
Through the application of a reference indicator, in the case of this method, in turn, disturbing influences can be eliminated. However, this method is only applicable for suitable combinations of luminescence-indicators, which can be excited with one and the same excitation wavelength and supplementally also possess a suitable relationship of the luminescence decay times, in order that a sufficient phase difference between the luminescence-signals is assured. Additionally, the measured signal is dependent on the concentration of the measuring, and the reference, indicator, or on their concentration ratio. In case one of the indicators is affected more strongly by leaching or bleaching, this leads to a degrading of the accuracy of measurement.